Dendrite morphogenesis in Caenorhabditis elegans.

Since the days of Ramón y Cajal, the vast diversity of neuronal and particularly dendrite morphology has been used to catalog neurons into different classes. Dendrite morphology varies greatly and reflects the different functions performed by different types of neurons. Significant progress has been made in our understanding of how dendrites form and the molecular factors and forces that shape these often elaborately sculpted structures. Here, we review work in the nematode Caenorhabditis elegans that has shed light on the developmental mechanisms that mediate dendrite morphogenesis with a focus on studies investigating ciliated sensory neurons and the highly elaborated dendritic trees of somatosensory neurons. These studies, which combine time-lapse imaging, genetics, and biochemistry, reveal an intricate network of factors that function both intrinsically in dendrites and extrinsically from surrounding tissues. Therefore, dendrite morphogenesis is the result of multiple tissue interactions, which ultimately determine the shape of dendritic arbors.

SAX-7/L1CAM acts with the adherens junction proteins MAGI-1, HMR-1/Cadherin, and AFD-1/Afadin to promote glial-mediated dendrite extension.

Adherens junctions (AJs) are a fundamental organizing structure for multicellular life. Although AJs are studied mainly in epithelia, their core function - stabilizing cell contacts by coupling adhesion molecules to the cytoskeleton - is important in diverse tissues. We find that two C. elegans sensory neurons, URX and BAG, require conserved AJ proteins for dendrite morphogenesis. We previously showed that URX and BAG dendrites attach to the embryonic nose via the adhesion molecule SAX-7/L1CAM, acting both in neurons and glia, and then extend by stretch during embryo elongation. Here, we find that a PDZ-binding motif (PB) in the SAX-7 cytoplasmic tail acts with other interaction motifs to promote dendrite extension. Using pull-down assays, we find that the SAX-7 PB binds the multi-PDZ scaffolding protein MAGI-1, which bridges it to the cadherin-catenin complex protein HMP-2/ß-catenin. Using cell-specific rescue and depletion, we find that both MAGI-1 and HMR-1/Cadherin act in glia to non-autonomously promote dendrite extension. Double mutant analysis indicates that each protein can act independently of SAX-7, suggesting a multivalent adhesion complex. The SAX-7 PB motif also binds AFD-1/Afadin, loss of which further enhances sax-7 BAG dendrite defects. As MAGI-1, HMR-1, and AFD-1 are all found in epithelial AJs, we propose that an AJ-like complex in glia promotes dendrite extension.

A sex-specific switch in a single glial cell patterns the apical extracellular matrix.

Apical extracellular matrix (aECM) constitutes the interface between every tissue and the outside world. It is patterned into diverse tissue-specific structures through unknown mechanisms. Here, we show that a male-specific genetic switch in a single C. elegans glial cell patterns the overlying aECM from a solid sheet to an ∼200 nm pore, thus allowing a male sensory neuron to access the environment. Using cell-specific genetic sex reversal, we find that this switch reflects an inherent sex difference in the glial cell that is independent of the sex identity of the surrounding neurons. Through candidate and unbiased genetic screens, we find that this glial sex difference is controlled by factors shared with neurons (mab-3, lep-2, and lep-5) as well as previously unidentified regulators whose effects may be glia specific (nfya-1, bed-3, and jmjd-3.1). The switch results in male-specific glial expression of a secreted Hedgehog-related protein, GRL-18, that we discover localizes to transient nanoscale rings at sites where aECM pores will form. Using electron microscopy, we find that blocking male-specific gene expression in glia prevents pore formation, whereas forcing male-specific glial gene expression induces an ectopic pore. Thus, a switch in gene expression in a single cell is necessary and sufficient to pattern aECM into a specific structure. Our results highlight that aECM is not a simple homogeneous meshwork, but instead is composed of discrete local features that reflect the identity of the underlying cells.

A sex-specific switch in a single glial cell patterns the apical extracellular matrix.

Apical extracellular matrix (aECM) constitutes the interface between every tissue and the outside world. It is patterned into diverse tissue-specific structures through unknown mechanisms. Here, we show that a male-specific genetic switch in a single C. elegans glial cell patterns the aECM into a ∻200 nm pore, allowing a male sensory neuron to access the environment. We find that this glial sex difference is controlled by factors shared with neurons ( mab-3, lep-2, lep-5 ) as well as previously unidentified regulators whose effects may be glia-specific ( nfya-1, bed-3, jmjd-3.1 ). The switch results in male-specific expression of a Hedgehog-related protein, GRL-18, that we discover localizes to transient nanoscale rings at sites of aECM pore formation. Blocking male-specific gene expression in glia prevents pore formation, whereas forcing male-specific expression induces an ectopic pore. Thus, a switch in gene expression in a single cell is necessary and sufficient to pattern aECM into a specific structure.

When is a neuron like an epithelial cell.

Neurons and epithelia are viewed as fundamentally different cell types, yet some sensory neurons exhibit hallmarks of epithelial cells. For example, they use tight junctions to form a diffusion barrier continuous with the skin or other epithelia and they exhibit bona fide apical-basal polarity, with an outward-facing apical surface that is biochemically and functionally distinct from their inward-facing basolateral surface. Yet they are unmistakeably neurons with axon-dendrite polarity. Examples include olfactory receptor neurons and photoreceptors. In this review, I highlight how viewing these neurons as specialized epithelial cells informs our understanding of their development and raises intriguing questions about the establishment of apical-basal and axon-dendrite polarity.

Axon-dendrite and apical-basolateral sorting in a single neuron.

Cells are highly organized machines with functionally specialized compartments. For example, membrane proteins are localized to axons or dendrites in neurons and to apical or basolateral surfaces in epithelial cells. Interestingly, many sensory cells-including vertebrate photoreceptors and olfactory neurons-exhibit both neuronal and epithelial features. Here, we show that Caenorhabditis elegans amphid neurons simultaneously exhibit axon-dendrite sorting like a neuron and apical-basolateral sorting like an epithelial cell. The distal ∼5-10 µm of the dendrite is apical, while the remainder of the dendrite, soma, and axon are basolateral. To determine how proteins are sorted among these compartments, we studied the localization of the conserved adhesion molecule SAX-7/L1CAM. Using minimal synthetic transmembrane proteins, we found that the 91-aa cytoplasmic tail of SAX-7 is necessary and sufficient to direct basolateral localization. Basolateral localization can be fully recapitulated using either of 2 short (10-aa or 19-aa) tail sequences that, respectively, resemble dileucine and Tyr-based motifs known to mediate sorting in mammalian epithelia. The Tyr-based motif is conserved in human L1CAM but had not previously been assigned a function. Disrupting key residues in either sequence leads to apical localization, while "improving" them to match epithelial sorting motifs leads to axon-only localization. Indeed, changing only 2 residues in a short motif is sufficient to redirect the protein between apical, basolateral, and axonal localization. Our results demonstrate that axon-dendrite and apical-basolateral sorting pathways can coexist in a single cell, and suggest that subtle changes to short sequence motifs are sufficient to redirect proteins between these pathways.

Loss of the Extracellular Matrix Protein DIG-1 Causes Glial Fragmentation, Dendrite Breakage, and Dendrite Extension Defects.

The extracellular matrix (ECM) guides and constrains the shape of the nervous system. In C. elegans, DIG-1 is a giant ECM component that is required for fasciculation of sensory dendrites during development and for maintenance of axon positions throughout life. We identified four novel alleles of dig-1 in three independent screens for mutants affecting disparate aspects of neuronal and glial morphogenesis. First, we find that disruption of DIG-1 causes fragmentation of the amphid sheath glial cell in larvae and young adults. Second, it causes severing of the BAG sensory dendrite from its terminus at the nose tip, apparently due to breakage of the dendrite as animals reach adulthood. Third, it causes embryonic defects in dendrite fasciculation in inner labial (IL2) sensory neurons, as previously reported, as well as rare defects in IL2 dendrite extension that are enhanced by loss of the apical ECM component DYF-7, suggesting that apical and basolateral ECM contribute separately to dendrite extension. Our results highlight novel roles for DIG-1 in maintaining the cellular integrity of neurons and glia, possibly by creating a barrier between structures in the nervous system.

Lineage-specific control of convergent differentiation by a Forkhead repressor.

During convergent differentiation, multiple developmental lineages produce a highly similar or identical cell type. However, few molecular players that drive convergent differentiation are known. Here, we show that the C. elegans Forkhead transcription factor UNC-130 is required in only one of three convergent lineages that produce the same glial cell type. UNC-130 acts transiently as a repressor in progenitors and newly-born terminal cells to allow the proper specification of cells related by lineage rather than by cell type or function. Specification defects correlate with UNC-130:DNA binding, and UNC-130 can be functionally replaced by its human homolog, the neural crest lineage determinant FoxD3. We propose that, in contrast to terminal selectors that activate cell type-specific transcriptional programs in terminally differentiating cells, UNC-130 acts early and specifically in one convergent lineage to produce a cell type that also arises from molecularly distinct progenitors in other lineages.

Cell-type-specific promoters for C. elegans glia.

Glia shape the development and function of the C. elegans nervous system, especially its sense organs and central neuropil (nerve ring). Cell-type-specific promoters allow investigators to label or manipulate individual glial cell types, and therefore provide a key tool for deciphering glial function. In this technical resource, we compare the specificity, brightness, and consistency of cell-type-specific promoters for C. elegans glia. We identify a set of promoters for the study of seven glial cell types (F16F9.3, amphid and phasmid sheath glia; F11C7.2, amphid sheath glia only; grl-2, amphid and phasmid socket glia; hlh-17, cephalic (CEP) sheath glia; and grl-18, inner labial (IL) socket glia) as well as a pan-glial promoter (mir-228). We compare these promoters to promoters that are expressed more variably in combinations of glial cell types (delm-1 and itx-1). We note that the expression of some promoters depends on external conditions or the internal state of the organism, such as developmental stage, suggesting glial plasticity. Finally, we demonstrate an approach for prospectively identifying cell-type-specific glial promoters using existing single-cell sequencing data, and we use this approach to identify two novel promoters specific to IL socket glia (col-53 and col-177).

Dendrites with specialized glial attachments develop by retrograde extension using SAX-7 and GRDN-1.

Dendrites develop elaborate morphologies in concert with surrounding glia, but the molecules that coordinate dendrite and glial morphogenesis are mostly unknown. C. elegans offers a powerful model for identifying such factors. Previous work in this system examined dendrites and glia that develop within epithelia, similar to mammalian sense organs. Here, we focus on the neurons BAG and URX, which are not part of an epithelium but instead form membranous attachments to a single glial cell at the nose, reminiscent of dendrite-glia contacts in the mammalian brain. We show that these dendrites develop by retrograde extension, in which the nascent dendrite endings anchor to the presumptive nose and then extend by stretching during embryo elongation. Using forward genetic screens, we find that dendrite development requires the adhesion protein SAX-7/L1CAM and the cytoplasmic protein GRDN-1/CCDC88C to anchor dendrite endings at the nose. SAX-7 acts in neurons and glia, while GRDN-1 acts in glia to non-autonomously promote dendrite extension. Thus, this work shows how glial factors can help to shape dendrites, and identifies a novel molecular mechanism for dendrite growth by retrograde extension.

Long-term activity drives dendritic branch elaboration of a C. elegans sensory neuron.

Neuronal activity often leads to alterations in gene expression and cellular architecture. The nematode Caenorhabditis elegans, owing to its compact translucent nervous system, is a powerful system in which to study conserved aspects of the development and plasticity of neuronal morphology. Here we focus on one pair of sensory neurons, termed URX, which the worm uses to sense and avoid high levels of environmental oxygen. Previous studies have reported that the URX neuron pair has variable branched endings at its dendritic sensory tip. By controlling oxygen levels and analyzing mutants, we found that these microtubule-rich branched endings grow over time as a consequence of neuronal activity in adulthood. We also find that the growth of these branches correlates with an increase in cellular sensitivity to particular ranges of oxygen that is observable in the behavior of older worms. Given the strengths of C. elegans as a model organism, URX may serve as a potent system for uncovering genes and mechanisms involved in activity-dependent morphological changes in neurons and possible adaptive changes in the aging nervous system.

A multicellular rosette-mediated collective dendrite extension.

Coordination of neurite morphogenesis with surrounding tissues is crucial to the establishment of neural circuits, but the underlying cellular and molecular mechanisms remain poorly understood. We show that neurons in a C. elegans sensory organ, called the amphid, undergo a collective dendrite extension to form the sensory nerve. The amphid neurons first assemble into a multicellular rosette. The vertex of the rosette, which becomes the dendrite tips, is attached to the anteriorly migrating epidermis and carried to the sensory depression, extruding the dendrites away from the neuronal cell bodies. Multiple adhesion molecules including DYF-7, SAX-7, HMR-1 and DLG-1 function redundantly in rosette-to-epidermis attachment. PAR-6 is localized to the rosette vertex and dendrite tips, and promotes DYF-7 localization and dendrite extension. Our results suggest a collective mechanism of neurite extension that is distinct from the classical pioneer-follower model and highlight the role of mechanical cues from surrounding tissues in shaping neurites.

Morphogenesis of neurons and glia within an epithelium.

To sense the outside world, some neurons protrude across epithelia, the cellular barriers that line every surface of our bodies. To study the morphogenesis of such neurons, we examined the C. elegans amphid, in which dendrites protrude through a glial channel at the nose. During development, amphid dendrites extend by attaching to the nose via DYF-7, a type of protein typically found in epithelial apical ECM. Here, we show that amphid neurons and glia exhibit epithelial properties, including tight junctions and apical-basal polarity, and develop in a manner resembling other epithelia. We find that DYF-7 is a fibril-forming apical ECM component that promotes formation of the tube-shaped glial channel, reminiscent of roles for apical ECM in other narrow epithelial tubes. We also identify a requirement for FRM-2, a homolog of EPBL15/moe/Yurt that promotes epithelial integrity in other systems. Finally, we show that other environmentally exposed neurons share a requirement for DYF-7. Together, our results suggest that these neurons and glia can be viewed as part of an epithelium continuous with the skin, and are shaped by mechanisms shared with other epithelia.

Ordered arrangement of dendrites within a C. elegans sensory nerve bundle.

Biological systems are organized into well-ordered structures and can evolve new patterns when perturbed. To identify principles underlying biological order, we turned to C. elegans for its simple anatomy and powerful genetics. We developed a method to quantify the arrangement of three dendrites in the main sensory nerve bundle, and found that they exhibit a stereotyped arrangement throughout larval growth. Dendrite order does not require prominent features including sensory cilia and glial junctions. In contrast, loss of the cell adhesion molecule (CAM) CDH-4/Fat-like cadherin causes dendrites to be ordered randomly, despite remaining bundled. Loss of the CAMs PTP-3/LAR or SAX-7/L1CAM causes dendrites to adopt an altered order, which becomes increasingly random as animals grow. Misexpression of SAX-7 leads to subtle but reproducible changes in dendrite order. Our results suggest that combinations of CAMs allow dendrites to self-organize into a stereotyped arrangement and can produce altered patterns when perturbed.

A neuronal MAP kinase constrains growth of a Caenorhabditis elegans sensory dendrite throughout the life of the organism.

Neurons develop elaborate morphologies that provide a model for understanding cellular architecture. By studying C. elegans sensory dendrites, we previously identified genes that act to promote the extension of ciliated sensory dendrites during embryogenesis. Interestingly, the nonciliated dendrite of the oxygen-sensing neuron URX is not affected by these genes, suggesting it develops through a distinct mechanism. Here, we use a visual forward genetic screen to identify mutants that affect URX dendrite morphogenesis. We find that disruption of the MAP kinase MAPK-15 or the ßH-spectrin SMA-1 causes a phenotype opposite to what we had seen before: dendrites extend normally during embryogenesis but begin to overgrow as the animals reach adulthood, ultimately extending up to 150% of their normal length. SMA-1 is broadly expressed and acts non-cell-autonomously, while MAPK-15 is expressed in many sensory neurons including URX and acts cell-autonomously. MAPK-15 acts at the time of overgrowth, localizes at the dendrite ending, and requires its kinase activity, suggesting it acts locally in time and space to constrain dendrite growth. Finally, we find that the oxygen-sensing guanylate cyclase GCY-35, which normally localizes at the dendrite ending, is localized throughout the overgrown region, and that overgrowth can be suppressed by overexpressing GCY-35 or by genetically mimicking elevated cGMP signaling. These results suggest that overgrowth may correspond to expansion of a sensory compartment at the dendrite ending, reminiscent of the remodeling of sensory cilia or dendritic spines. Thus, in contrast to established pathways that promote dendrite growth during early development, our results reveal a distinct mechanism that constrains dendrite growth throughout the life of the animal, possibly by controlling the size of a sensory compartment at the dendrite ending.

Coordinated morphogenesis of neurons and glia.

Glia adopt remarkable shapes that are tightly coordinated with the morphologies of their neuronal partners. To achieve these precise shapes, glia and neurons exhibit coordinated morphological changes on the time scale of minutes and on size scales ranging from nanometers to hundreds of microns. Here, we review recent studies that reveal the highly dynamic, localized morphological changes of mammalian neuron-glia contacts. We then explore the power of Drosophila and C. elegans models to study coordinated changes at defined neuron-glia contacts, highlighting the use of innovative genetic and imaging tools to uncover the molecular mechanisms responsible for coordinated morphogenesis of neurons and glia.

A Conserved Role for Girdin in Basal Body Positioning and Ciliogenesis.

Primary cilia are ubiquitous sensory organelles that mediate diverse signaling pathways. Cilia position on the cell surface is determined by the location of the basal body (BB) that templates the cilium. The mechanisms that regulate BB positioning in the context of ciliogenesis are largely unknown. Here we show that the conserved signaling and scaffolding protein Girdin localizes to the proximal regions of centrioles and regulates BB positioning and ciliogenesis in Caenorhabditis elegans sensory neurons and human RPE-1 cells. Girdin depletion alters localization of the intercentriolar linker and ciliary rootlet component rootletin, and rootletin knockdown in RPE-1 cells mimics Girdin-dependent phenotypes. C. elegans Girdin also regulates localization of the apical junction component AJM-1, suggesting that in nematodes Girdin may position BBs via rootletin- and AJM-1-dependent anchoring to the cytoskeleton and plasma membrane, respectively. Together, our results describe a conserved role for Girdin in BB positioning and ciliogenesis.

Duplication of a Single Neuron in C. elegans Reveals a Pathway for Dendrite Tiling by Mutual Repulsion.

Simple cell-cell interactions can give rise to complex cellular patterns. For example, neurons of the same type can interact to create a complex patchwork of non-overlapping dendrite arbors, a pattern known as dendrite tiling. Dendrite tiling often involves mutual repulsion between neighboring neurons. While dendrite tiling is found across nervous systems, the nematode Caenorhabditis elegans has a relatively simple nervous system with few opportunities for tiling. Here, we show that genetic duplication of a single neuron, PVD, is sufficient to create dendrite tiling among the resulting ectopic neurons. We use laser ablation to show that this tiling is mediated by mutual repulsion between neighbors. Furthermore, we find that tiling requires a repulsion signal (UNC-6/Netrin and its receptors UNC-40/DCC and UNC-5) that normally patterns the PVD dendrite arbor. These results demonstrate that an apparently complex cellular pattern can emerge in a simple nervous system merely by increasing neuron number.

Computer-Assisted Transgenesis of Caenorhabditis elegans for Deep Phenotyping.

A major goal in the study of human diseases is to assign functions to genes or genetic variants. The model organism Caenorhabditis elegans provides a powerful tool because homologs of many human genes are identifiable, and large collections of genetic vectors and mutant strains are available. However, the delivery of such vector libraries into mutant strains remains a long-standing experimental bottleneck for phenotypic analysis. Here, we present a computer-assisted microinjection platform to streamline the production of transgenic C. elegans with multiple vectors for deep phenotyping. Briefly, animals are immobilized in a temperature-sensitive hydrogel using a standard multiwell platform. Microinjections are then performed under control of an automated microscope using precision robotics driven by customized computer vision algorithms. We demonstrate utility by phenotyping the morphology of 12 neuronal classes in six mutant backgrounds using combinations of neuron-type-specific fluorescent reporters. This technology can industrialize the assignment of in vivo gene function by enabling large-scale transgenic engineering.

FBN-1, a fibrillin-related protein, is required for resistance of the epidermis to mechanical deformation during C. elegans embryogenesis.

During development, biomechanical forces contour the body and provide shape to internal organs. Using genetic and molecular approaches in combination with a FRET-based tension sensor, we characterized a pulling force exerted by the elongating pharynx (foregut) on the anterior epidermis during C. elegans embryogenesis. Resistance of the epidermis to this force and to actomyosin-based circumferential constricting forces is mediated by FBN-1, a ZP domain protein related to vertebrate fibrillins. fbn-1 was required specifically within the epidermis and FBN-1 was expressed in epidermal cells and secreted to the apical surface as a putative component of the embryonic sheath. Tiling array studies indicated that fbn-1 mRNA processing requires the conserved alternative splicing factor MEC-8/RBPMS. The conserved SYM-3/FAM102A and SYM-4/WDR44 proteins, which are linked to protein trafficking, function as additional components of this network. Our studies demonstrate the importance of the apical extracellular matrix in preventing mechanical deformation of the epidermis during development.